A polypeptide conjugate targeting m1 macrophages and uses thereof

Abstract

The application discloses a polypeptide conjugated drug targeting M1 type macrophages and application thereof, and belongs to the technical field of polypeptide conjugated drugs. The polypeptide conjugated drug M1pep-Tasquinimod is composed of a polypeptide targeting M1 type macrophages, a linker and a modified Tasquinimod. The polypeptide conjugated drug can reduce the toxicity of the Tasquinimod, inhibit M1 type polarization of macrophages by targeting proinflammatory M1 type macrophages, and relieve pancreatic tissue lesions of acute pancreatitis mice. The application provides a basis for using the M1pep-Tasquinimod as a novel drug for treating inflammatory diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptide conjugate drugs, and particularly relates to a polypeptide conjugate drug targeting M1 type macrophages and application thereof. BACKGROUND

[0002] Acute pancreatitis (AP) is a common digestive system disease. About 20% of AP patients develop into severe pancreatitis, and the mortality rate is more than 30%. The imbalance between pro-inflammatory cytokines and anti-inflammatory cytokine responses causes the disorder of the body's immune regulation system, induces the inflammatory cascade, and is a key factor for the severity of AP. Macrophages, as the first line of defense of the human body, play an important role in maintaining the homeostasis of the internal environment and the inflammatory microenvironment. In recent years, macrophage polarization has attracted widespread attention in the scientific community, and a variety of inflammation-related diseases have been confirmed to be related to macrophage polarization. Mature macrophages have high plasticity and are mainly divided into: classical activated (M1) type with pro-inflammatory function and alternative activated (M2) type with anti-inflammatory phenotype according to different surface antigens and functions. Interferon-gamma, lipopolysaccharide (LPS), granulocyte / macrophage colony-stimulating factor (G / M-CSF) or other toll-like receptor (TLR) ligands stimulate can promote the polarization of M1 type macrophages, and the polarized macrophages express a large number of pro-inflammatory cytokines (such as TNF-α, IL-1β and IL-6) and specific chemokines (such as CCL2-5, CCL8, CXCL-2, CXCL-4 and CXCL-9), induce Th1 type cellular immune response, and are an important driving factor for the severity of AP. Therefore, drug therapy targeting M1 type macrophages may become an important intervention measure for the inflammatory cascade.

[0003] S100A9 is a calcium-binding protein, mainly expressed in monocyte-macrophage system, with anti-inflammatory and pro-inflammatory dual effects. Our previous study found that S100A9 plays an important role in the development of pancreatitis. S100A9 binds to TLR4 to activate NF-kB, which can induce the pro-inflammatory function of macrophages and inhibit the polarization of M2 type macrophages. Tasquinimod (CAS No: 254964-60-8) is an orally active quinoline-3-carboxamide, which is currently being studied as an experimental drug for the treatment of solid tumors. Tasquinimod, as a small molecule oral inhibitor of S100A9, can inhibit the interaction between S100A9 and its late glycosylation end product ligand receptor and Toll-like receptor 4. However, Tasquinimod has certain toxicity, and long-term large-dose consumption may cause irreversible harm to the human body. The success of antibody conjugated drugs (ADC) has opened the huge screen of the "era of everything conjugation". Compared with ADC drugs, polypeptide conjugated drugs (PDC) have the advantages of small molecular weight, strong penetration, low immunogenicity and low production cost, and are expected to become a new generation of targeted drugs after small molecule drugs, monoclonal antibodies and ADC drugs. Current PDC research focuses on targeted anti-tumor drugs, and there is no related research on PDC targeting M1 type macrophages for the treatment of inflammatory diseases. SUMMARY

[0004] In view of this, the purpose of the present application is to provide a polypeptide conjugated drug targeting M1 type macrophages and its application, which can inhibit the polarization of M1 type macrophages by targeting pro-inflammatory M1 type macrophages, and reduce sodium taurocholate-induced acute pancreatitis in mice.

[0005] The purpose of the present application is achieved by the following means:

[0006] The present application provides a polypeptide conjugated drug targeting M1 type macrophages, which is composed of a polypeptide targeting M1 type macrophages, a linker and a modified Tasquinimod.

[0007] Based on the above technical solution, further, the polypeptide is FSDDCYDCRIPR or VHAVPIRTIYYP.

[0008] Based on the above technical solution, further, the amino acid sequence of the linker is GGGSKKK.

[0009] Based on the above technical solution, further, the structure of the modified Tasquinimod is as follows:

[0010]

[0011] Based on the above technical scheme, further, the structure of the polypeptide conjugated drug is as follows:

[0012]

[0013]

[0014] Another aspect of the present application provides a pharmaceutical composition comprising an effective amount of the above polypeptide conjugated drug and a pharmaceutically acceptable carrier.

[0015] Based on the above technical scheme, further, the pharmaceutically acceptable carrier comprises a filler, a diluent, a binder, a disintegrant, an emulsifier and a drug carrier with no toxic side effects.

[0016] The present application also provides the use of the above polypeptide conjugated drug and the pharmaceutical composition in the preparation of a drug for improving acute pancreatitis.

[0017] Based on the above technical scheme, further, the dosage form of the drug comprises tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

[0018] The present application has the following beneficial effects relative to the prior art:

[0019] The present application adopts the polypeptide conjugated drug M1pep-Tasquinimod targeting M1 type macrophages to inhibit M1 type macrophage polarization in vitro and treat acute pancreatitis mouse models in vivo, which proves that M1pep-Tasquinimod can inhibit LPS-induced M1 type macrophage polarization and reduce the pancreatic tissue damage of acute pancreatitis mice, and the present application provides a basis for using M1pep-Tasquinimod as a new drug for treating inflammatory diseases. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0021] Figure 1 (a) is FSDDCYDCRIPRGGGSKK-[K modified Tasquinimod], (b) is VHAVPIRTIYYPGGGSKK-[K modified Tasquinimod], and (c) is WEDYQWPVYKGWGGGSKK-[K modified Tasquinimod].

[0022] Figure 2 is a flow cytometry detection of M1 type polarization of macrophages in different experimental groups, compared with the LPS group,

[0023] **P<0.01; compared with Tasquinimod group, ## P<0.01.

[0024] Figure 3 For qPCR detection of M1 macrophage markers in different experimental groups, compared with LPS group,

[0025] **P<0.01; compared with Tasquinimod group, # P<0.05, ## P<0.01.

[0026] Figure 4 For the results of toxicity detection of mice after administration, (a) is the observation of pathological damage of heart, liver, spleen, lung, kidney, pancreas and intestinal tissue by HE staining, (b) is the content of creatinine (Cre), urea nitrogen (Bun), ALT and AST in serum.

[0027] Figure 5 For the results of HE staining of pancreas (a, image magnification 200 times) and histopathology score (b) of mice after administration and treatment, compared with AP group, **P<0.01; compared with Tasquinimod-15 group, ##P<0.01.

[0028] Figure 6 For the results of serum amylase and lipase detection of mice after administration and treatment, compared with AP group, *P<0.01, **P<0.01; compared with Tasquinimod-15 group, ## P<0.01. DETAILED DESCRIPTION

[0029] The application will be described in detail below with reference to the examples, but the embodiments of the application are not limited thereto. Obviously, the examples described below are only part of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0030] Example 1

[0031] 1. Tasquinimod structure modification, the reaction formula is as follows:

[0032]

[0033] including the following steps:

[0034] (1) In a single neck flask, concentrated hydrochloric acid (3 mL) of 38% mass fraction was slowly added into glacial acetic acid (9 mL), then compound 1 Cas: 248282-13-5 (1 g, 1 eq) was added and heated to 70 °C for 18 hours, LCMS monitored product formation, raw material disappeared, the reaction was cooled to room temperature. The formed solid was filtered and washed with ethanol (20 mL). White solid compound 2 (0.6 g, 66% yield) was obtained by drying;

[0035] (2) In a single neck flask, HATU (1.83 g, 2 eq), DIPEA (0.94 g, 3 eq) were added into a solution of compound 2 (0.6 g, 1 eq) in DMF (10 mL) / THF (10 mL) in turn, stirred at room temperature for 10 min, then compound 2a (0.48 g, 1.2 eq) was added and stirred at 50 °C for 2 hours. LCMS monitored product formation, raw material disappeared. The pH was adjusted to 6-7 by adding dilute hydrochloric acid, concentrated, purified by 4.6*150 mm, kromasil C18-5 liquid chromatography column (20%-55% MeCN, 0.1% TFA), concentrated, freeze-dried to obtain yellow oil compound 3 (0.5 g, 52.6% yield);

[0036] (3) In a single neck flask, 5 mL aqueous solution of KOH (0.21 g, 3 eq) was added to a solution of compound 3 (0.5 g, 1 eq) in THF (10 mL), stirred at 50 °C for 4 hours. LCMS monitored product formation, raw material disappeared, the pH was adjusted to 5-6 by adding dilute hydrochloric acid, concentrated, poured out the water, the bottom yellow viscous material was ultrasonicated with 5 mL anhydrous methanol, filtered, the filter solid was dried to obtain white solid compound 4 (0.2 g, 41.5% yield);

[0037] (4) DCC (0.13 g, 1.2 eq) was added to a suspension of compound 4 (0.2 g, 1 eq) in DMF (5 mL), then compound 4a (90 mg, 1.5 eq), DMAP (6.4 mg, 0.1 eq) were added in turn, stirred at 50 °C for 2 hours. Filtration, the filtrate was purified by 4.6*150 mm, kromasil C18-5 liquid chromatography column (20%-50% MeCN, 0.1% TFA), concentrated and freeze-dried to obtain white solid modified tasquinimod (0.12 g, 48% yield, 97.8% purity, M+H+=480.1).

[0038] 2. Chemical synthesis of M1pep-tasquinimod

[0039] (1) Resin swelling: Weigh the resin and put it into the reaction column, then add DCM into the reaction column and shake for 30 min. (2) Link the first amino acid: Filter out the DCM solvent by suction, add the first amino acid, then add 10 times the molar amount of DIEA, and finally add a small amount of DMF to dissolve and shake for 1 hour. After the reaction is complete, wash the reaction with DMF and DCM alternately for 6 times. (3) Deprotection: Add 20% piperidine / DMF solution, after 5 min, suction, then add 20% piperidine / DMF solution and shake for 15 min. (4) Detection: Suction the piperidine solution, take a dozen resin particles and wash with ethanol for three times, add one drop of ninhydrin, pyridine and phenol respectively, heat at 105-110°C for 5 min, dark blue is positive, if no color change, it is negative, and needs to be re-deprotected. (5) First washing: Wash with DMF, methanol and DMF respectively for two times. (6) Condensation: Add the amino acid with a protective group, condensing agent HBTU, a small amount of DMF to dissolve, immediately add DIEA, and react for 30 min. (7) Second washing: Wash with DMF, methanol and DMF respectively for two times. (8) Detection: Suction the solvent, take a dozen resin particles and wash with ethanol for three times, add one drop of ninhydrin, pyridine and phenol respectively, heat at 105-110°C for 5 min, colorless is positive, if blue, it needs to be re-condensed. (9) Extension of the peptide chain: Repeat the above operations in turn to add the remaining amino acids. (10) Link the activated small molecule: Add 2% hydrazine hydrate / DMF solution, after 5 min, suction, repeat for 2 times, add the activated molecule of the modified Tasquinimod, a small amount of DMF to dissolve, immediately add DIEA, and react for 30 min. (11) Detection: Suction the solvent, take a dozen resin particles and wash with ethanol for three times, add one drop of ninhydrin, pyridine and phenol respectively, heat at 105-110°C for 5 min, colorless is positive, if blue, it needs to be re-condensed. (12) Peptide chain contraction: After the last amino acid is added, the synthesis of the whole peptide is completed, and the final contraction stage is entered. Wash the reaction with DMF for 3 times, DCM for 3 times, and methanol for 3 times, and then suction the peptide resin to dry. (13) Amino acid side chain deprotection and resin cleavage: Prepare 15 mL of cleavage solution, and the volume ratio of each component is: TFA (94.5%), water (2%), EDT (2.5%), and TIS (1%). Put the resin into a flask and shake at constant temperature (30°C) for 2 hours. Blow the cleavage solution to dryness with nitrogen as much as possible, then pour it into a centrifuge tube, and slowly pour into ethyl ether. Seal and put into the centrifuge for 5 min, pour off the supernatant, and the white solid is at the bottom. Wash with ethyl ether for 6 times and then dry at room temperature to obtain the crude peptide.

[0040] This example obtains three kinds of crude peptide samples, and the specific structures are as shown in Figure 1 ​

[0041] FSDDCYDCRIPRGGGSKK-[K remodeled Tasquinimod], i.e. M1pep1-Tasquinimod; (b) is VHAVPIRTIYYPGGGSKK-[K remodeled Tasquinimod], i.e. M1pep10-Tasquinimod; (c) is WEDYQWPVYKGWGGGSKK-[K remodeled Tasquinimod] (control drug), i.e. NCpep-Tasquinimod.

[0042] Example 2

[0043] This example is an in vitro experiment. Raw264.7 cells were inoculated in a 6-well plate and cultured for 24 h, and were divided into a control group, a model group, a drug treatment group (divided into a high-dose group and a low-dose group, each dose group including a Taquinimod group, a M1pep1-Tasquinimod group, a M1pep10-Tasquinimod group and a NCpep-Tasquinimod group), medium was added in the control group, 1 μg / mL LPS + 20 ng / mL IFN-γ medium was added in the model group and the drug treatment group, and incubated for 24 h. The culture medium was aspirated, and the culture medium was added. In the treatment group, 20 μM Taquinimod, M1pep1-Tasquinimod, M1pep10-Tasquinimod and NCpep-Tasquinimod were added, respectively, and incubated in an incubator for 24 h. The cell precipitate was collected for subsequent flow cytometry detection and qPCR experiments.

[0044] Flow cytometry detection: collect cells in each group in a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash once with PBS, and detect on the machine.

[0045] qPCR experiment: trizol was used to extract RNA from each group of cells, reverse transcription was performed according to the instructions of the All-in-One First-Strand Synthesis MasterMix (with dsDNase) kit to obtain cDNA, and Taq-HS Green qPCR Premix(Universal) kit to prepare the qPCR system, and detect on the machine.

[0046] The results of the in vitro experiment are shown in Figures 2-3 LPS+IFN-γ group Figure 2Compared with the LPS group, the Tasquinimod group can significantly reduce the proportion of M1 polarization of Raw264.7 cells, inhibit the expression of M1 macrophage markers iNOS, CD86 and TNF-α genes, and the M1pep-Tasquinimod group has a better effect on inhibiting the polarization of M1 macrophages than the Tasquinimod group, while the NCpep-Tasquinimod group has no obvious change compared with the Tasquinimod group, indicating that M1pep-Tasquinimod enhances the pharmacodynamic effect of Tasquinimod.

[0047] Example 3

[0048] This example is a mouse in vivo experiment. According to the method reported in the literature (doi: 10.1038 / nprot.2009.243), the acute pancreatitis mouse model was induced by retrograde injection of sodium taurocholate into the pancreatic duct. 54 C57BL / 6 mice were randomly divided into a model group, a drug treatment group (divided into a high-dose group and a low-dose group, each dose group including a Taquinimod group, a M1pep1-Tasquinimod group, a M1pep10-Tasquinimod group and a NCpep-Tasquinimod group), and 6 mice in each group. The specific operation is as follows: the mice were fasted for 12 hours before the experiment, and anesthetized by isoflurane inhalation. After successful anesthesia, the mice were placed in a supine position, and the head and limbs were fixed. Under sterile operation, an abdominal incision was made in the middle of the upper abdomen, the operative field was fully exposed, the duodenum was taken out and flattened, the bile duct near the hepatic portal was clamped to prevent drug injection into the bile duct, a 31G needle (diameter 0.25mm) was connected to the injection pump, and the operation was performed under a small animal microscope. The needle was inserted into the pancreaticobiliary junction, and 30μL of 5% sodium taurocholate solution was slowly injected retrograde. After injection, the needle was withdrawn, the pancreas was carefully replaced, and the abdomen was sutured layer by layer. After the operation, attention should be paid to keep warm, fasting and free drinking water. The control group was opened in the same way, and the same amount of sterile normal saline was injected retrograde into the pancreaticobiliary duct. The drug treatment group was injected intraperitoneally with Taquinimod (15mg / kg, 30mg / kg), M1pep1-Tasquinimod: FSDDCYDCRIPRGGGSKK-[K modified Tasquinimod] (15mg / kg, 30mg / kg), M1pep10-Tasquinimod: VHAVPIRTIYYPGGGSKK-[K modified Tasquinimod] (15mg / kg, 30mg / kg), NCpep-Tasquinimod:

[0049] WEDYQWPVYKGWGGGSKK-[K remodeled Tasquinimod] (15 mg / kg, 30 mg / kg); the model group was given an equal volume of normal saline. At 24 h after administration, the mice were anesthetized by isoflurane inhalation, serum was collected, and stored at -80°C. The pancreas, heart, liver, spleen, lung, kidney, and part of the intestinal tissue were placed in paraformaldehyde tissue fixative for HE staining to observe the histopathological changes.

[0050] The pathological damage of the heart, liver, spleen, lung, kidney, and intestinal tissue was observed by HE staining, the serum amylase and lipase levels were detected by an amylase and lipase assay kit, and the serum Cre, Bun, ALT, and AST contents were detected by a Cre, Bun, ALT, and AST assay kit.

[0051] The results of the toxicity experiment are shown in Table 2. Figure 4 Compared with the model group, the Taquinimod group (15 mg / kg, 30 mg / kg) and the NCpep-Tasquinimod group (15 mg / kg, 30 mg / kg) showed glomerular pyknosis and structural damage, suggesting that they had nephrotoxicity, while the M1pep1-Tasquinimod group and the M1pep10-Tasquinimod group showed glomerular damage only at a dose of 30 mg / kg. Compared with the model group, the Taquinimod group (30 mg / kg) and the NCpep-Tasquinimod group (30 mg / kg) showed an increase in immune cells in the spleen, immune hyperactivity, and a significantly higher Bun content.

[0052] The pathological damage of the pancreas tissue was observed by HE staining, and the results are shown in Table 3. Figure 5 Compared with the model group, the Tasquinimod group (15 mg / kg, 30 mg / kg) showed reduced edema, hemorrhage, acinar cell necrosis, and inflammatory cell infiltration in the mouse pancreas tissue, and compared with the Tasquinimod group (15 mg / kg, 30 mg / kg), the M1pep-Tasquinimod group (15 mg / kg, 30 mg / kg) showed more obvious improvement in the damage to the mouse pancreas tissue, while the NCpep-Tasquinimod group (15 mg / kg, 30 mg / kg) showed no significant change compared with the Tasquinimod group. The above results show that Taquinimod has an improving effect on acute pancreatitis in mice, and M1pep-Tasquinimod has a better therapeutic effect than Taquinimod alone.

[0053] The detection results of serum amylase and lipase are shown in Table 4. Figure 6As shown, compared with the model group, the serum amylase and lipase levels of the Tasquinimod group (15 mg / kg, 30 mg / kg), the M1pep-Tasquinimod group (15 mg / kg, 30 mg / kg), and the NCpep-Tasquinimod group (15 mg / kg, 30 mg / kg) mice were significantly reduced.

[0054] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A polypeptide-conjugated drug targeting M1 macrophages, characterized in that: The structure of the peptide-coupled drug is shown below:

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the polypeptide-coupled drug according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutically acceptable carrier includes a filler, a diluent, a binder, a disintegrant and an emulsifier.

4. Use of the polypeptide-coupled drug according to claim 1 or the pharmaceutical composition according to claim 2 or 3 in the preparation of a medicament for improving acute pancreatitis.

5. The use according to claim 4, characterized in that The dosage forms of the medicine include tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

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